System and method for dynamic chemical imaging

ABSTRACT

A system and method for detecting dynamic changes that occur in a sample between a first time interval and a second time interval using a series of at least first and second sequential chemical images of the sample. During the first time interval: (i) the sample is illuminated with a plurality of photons to thereby produce photons scattered or emitted by the sample; (ii) a two-dimensional array of detection elements is used to simultaneously detect scattered or emitted photons in a first predetermined wavelength band from different locations on or within the sample; and (iii) the two-dimensional array of detection elements is thereafter used one or more further times to simultaneously detect scattered or emitted photons in one or more further predetermined wavelength band(s) from different locations on or within the sample. The outputs of the two-dimensional array of detection elements during the first time interval are then combined to generate the first chemical image of the sample. The process is repeated during the second time interval to generate the second chemical image of the sample. Dynamic changes occurring in the sample between the first time interval and the second time interval are detected based on one or more differences between the first and second chemical images.

RELATED APPLICATIONS

The present application claims the filing-date benefit of applicationSer. No. 10/698,243 filed Oct. 31, 2003 and Ser. No. 10/698,584 filedOct. 31, 2003, as well as provisional application No. 60/422,604 filedOct. 31, 2002, each of which is incorporated herein by reference in itsentirety. The present application is also related to U.S. patentapplication Ser. No. ______, filed contemporaneously herewith, entitled“Method And Apparatus For Dark Field Chemical Imaging,” alsoincorporated herein by reference in its entirety. Each of theabove-referenced applications is assigned to the assignee of the presentapplication.

FIELD OF THE INVENTION

The present invention relates generally to chemical imaging and, inparticular, to the use of chemical imaging to detect dynamic changes ina sample.

BACKGROUND

Chemical imaging is known in the art. One example of an apparatus usedfor chemical imaging is taught in U.S. Pat. No. 6,002,476, entitled“Chemical Imaging System,” to Treado et al. Among other things, U.S.Pat. No. 6,002,476 teaches the use of Raman chemical imaging foranalysis of a static sample, e.g., for assessing whether a particulartissue sample corresponds to normal tissue or breast cancer tissue.Other chemical imaging systems for assessment of static samples exist inthe art.

In contrast to the prior art, the present invention uses chemicalimaging to assess and observe non-static samples (i.e., samples thatvary over time). Among other things, the present invention may be usedto detect dynamic changes that occur in the sample over an observationperiod.

SUMMARY OF THE INVENTION

The present invention is directed to a system and method for detectingdynamic changes that occur in a sample between a first time interval anda second time interval using a series of at least first and secondsequential chemical images of the sample, wherein the first chemicalimage corresponds to an image of the sample during a first timeinterval, and the second chemical image corresponds to an image of thesample at a second time interval after the first time interval. Duringthe first time interval: (i) the sample is illuminated with a pluralityof photons to thereby produce photons scattered or emitted by thesample; (ii) a two-dimensional array of detection elements is then usedto simultaneously detect scattered or emitted photons in a firstpredetermined wavelength band from different locations on or within thesample; and (iii) the two-dimensional array of detection elements isthereafter used one or more further times to simultaneously detectscattered or emitted photons in one or more further predeterminedwavelength band(s) from different locations on or within the sample. Theoutputs of the two-dimensional array of detection elements during thefirst time interval are then combined to generate the first chemicalimage of the sample.

During the second time interval: (i) the sample is illuminated with aplurality of photons to thereby produce photons scattered or emitted bythe sample; (ii) the two-dimensional array of detection elements is thenused to simultaneously detect scattered or emitted photons in a firstpredetermined wavelength band from different locations on or within thesample; and (iii) the two-dimensional array of detection elements isthereafter used one or more further times to simultaneously detectscattered or emitted photons in one or more further predeterminedwavelength band(s) from different locations on or within the sample. Theoutputs of the two-dimensional array of detection elements during thesecond time interval are then combined to generate the second chemicalimage of the sample.

Dynamic changes occurring in the sample between the first time intervaland the second time interval are next detected based on one or moredifferences between the first and second chemical images.

The present invention permits rapid observation of the sample with fullspatial information, and allows the monitoring of the evolution andchanges in the sample that are naturally proceeding or occurring (i.e.,under equilibrium conditions,) as well as those that are additionallyforced or imposed by creating a non-equilibrium condition via anexternal means (e.g., one or more external fields or forces applied tothe sample). In certain embodiments, the external means may be appliedto a specific location within the sample (rather than the whole sample).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically represents an apparatus according to one embodimentof the disclosure; and

FIG. 2 schematically represent an apparatus according to anotherembodiment of the disclosure.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

FIG. 1 schematically represents an apparatus according to one embodimentof the disclosure. The apparatus of FIG. 1 enables providing a highoptical throughput for imaging low light levels at variablemagnification. Referring to FIG. 1, sample 100 is positioned onsubstrate 105. Substrate 105 can be any conventional microscopic slideor other means for receiving and optionally securing sample 100. Lightsource 110 is positioned to provide incident light to sample 100. Lightsource 110 can include any conventional photon source, including laser,LED, and other IR or near IR devices. Light source 110 may also beselected to provide evanescence illumination of the sample. In oneembodiment, the wavelength of the source is in the range of about 15-25cm⁻¹.

Referring still to FIG. 1, it should be noted that light source 110 ispositioned to provide incident light at an angle to sample 100 asopposed to light shining orthogonal to sample 100. In other words, theradiation used to illuminate the sample need not pass through theoptical train of a conventional microscope (or macroscope); rather, itcan illuminate the sample at an oblique angle from above or below sample100. Photon beam 112 is received and deflected by mirror 115 throughlens 120. Lens 120 may optionally be used to focus the light on sample100. Alternatively, the photon beam 112 may be directed towards thesample 100 without the need for the mirror 115.

The multitude of photons in beam 112 reaching sample 100 illuminate thesample and are either scattered or absorbed by the sample, which canresult in subsequent emission (luminescence) at different wavelengths.As known to those skilled in the art, the term “luminescence” includes awide range of optical processes described using other names. Theseinclude: fluorescence, phosporescence, photoluminescence,electroluminescence, chemiluminescence, sonoluminescence,thermoluminescence and even upconversion. Scattered photons areschematically represented as beams 116 and 118 while specularlyreflected photons are represented schematically as beam 114.Luminescence emitted photons are also represented as beam 118. Opticallens 125 is positioned to receive photon beams 116 and 118. Optical lens125 may be used for gathering and focusing received photon beams. Thisincludes gathering and focusing both polarized and the un-polarizedphotons. In general, the sample size determines the choice of lightgathering optical lens 125. For example, a microscope lens may beemployed for analysis of the sub-micron to micrometer specimens. Forlarger samples, macro lenses can be used. Optical lens 125 (as well aslens 120) may include a simple reduced resolution/aberration lens with alarger numerical aperture to thereby increase system's opticalthroughput and efficiency. Mirror 130 is positioned to direct emitted orscattered photon beams 118 to tunable filter 140. It should be notedthat placement of mirror 130 is optional and may be unnecessary inconfigurations where tunable filter is positioned above sample 100.

Laser rejection filter 135 may be positioned prior to tunable filter 140to filter out scattered illumination light represented by beam 116 andto optimize the performance of the system. In other words, rejectionfilter 135 enables spectrally filtering of the photons at theilluminating wavelength.

A conventional tunable filter (including electro-optical tunablefilters) including an liquid crystal tunable filter (“LCTF”) oracousto-optical tunable filter (“AOTF”) can be used to further theprinciples of the disclosure. The electro-optical filters(interchangeably, tunable filters) allow specific wavelengths or rangesof wavelengths of light to pass through as an image, depending on thecontrol signals placed on the device by a controller (not shown). Thewavelengths that can be passed through tunable filter 140 may range from200 nm (ultraviolet) to 2000 nm (i.e., the far infrared). The choice ofwavelength depends on the desired optical region and/or the nature ofthe sample being analyzed.

Image sensor 145 may be a digital device such as for example atwo-dimensional, image focal plane array (“FPA”) or CCD or CMOS sensor.The optical region employed to characterize the sample of interestgoverns the choice of FPA detector. For example, a two-dimensional arrayof silicon charge-coupled device (“CCD”) detection elements, can beemployed with visible wavelength fluorescence and Raman spectroscopic,while gallium arsenide (GaAs) and gallium indium arsenide (GaInAs) FPAdetectors can be employed for image analyses at near infraredwavelengths. The choice of such devices depends on the type of samplebeing analyzed. In one embodiment, each detection element in thetwo-dimensional array of detection elements used to form image sensor145 functions to detect photons scattered or emitted from a differentspatial location on or within the sample. In one embodiment, imagesensor 145 produces digital images of the entire view of the sample asprocessed by tunable filter 140.

FIG. 2 schematically represents an apparatus according to anotherembodiment of the disclosure. More specifically, FIG. 2 schematicallyshows a high optical throughput configuration for imaging low lightlevels at variable magnification. The collection of optics are similarto that illustrated in FIG. 1 but with illumination from the undersideof sample 100.

It is noted that in both FIGS. 1 and 2, sample 100 is illuminated at anoblique angle. Specifically referring to FIG. 2, photonic beam 120 andthe plane axis of sample 100 define an oblique angle. It has been foundthat through oblique illumination, a so-called “Dark Field RamanImaging” is developed. As opposed to the conventional bright field Ramanconfiguration, the dark field Raman imaging decouples the image captureoptics from the deliver of exciting radiation. Consequently, internalscattering and attenuation of the incident radiation has been minimizedto improve the signal to noise ratio. Also, the location of the opticalsource external to the optical train further allows the use of a lowercost, less powerful illumination source as well as enables a simpler,less expensive integration of several illumination sources into thesystem. The application of this configuration is not limited to Ramanand luminescence imaging and can be successfully used, for example, withconventional spectroscopy.

In each of the embodiments shown in FIGS. 1 and 2, a computer orprocessor (not shown) is coupled to and used to control the opticaldevices including light source (110), lenses (120, 125, 135), mirrors(115, 130) and tunable filter (140). The computer is also coupled toimage sensor 145 and functions to generate “chemical images” from theoutput of the image sensor 145. In one embodiment, each chemical imageis a spatially accurate wavelength-resolved image of the sample that isformed from multiple “frames”; wherein each frame has plural spatialdimensions and is created from photons of a particular wavelength (orwave number) or from photons in a particular wavelength band (or wavenumber band) that are collected simultaneously by image sensor 145 fromdifferent spatial locations on or within sample 100. In each chemicalimage, multiple frames may be combined to form a complete image acrossall wavelengths (wave numbers) of interest. The chemical imagesgenerated by the computer may be further analyzed by the computer and/ordisplayed to a user.

The present invention uses an apparatus such as those shown in FIGS. 1and 2 to detect dynamic changes that occur in sample 100 between a firsttime interval and a second subsequent time interval using a series of atleast first and second sequential chemical images of sample 100. Duringthe first time interval: (i) sample 100 is illuminated with photons fromsource 110 to thereby produce photons scattered or emitted by sample100; (ii) image sensor 145 is then used to simultaneously detectscattered or emitted photons in a first predetermined wavelength band(selected by tunable filter 140) from different locations on or withinthe sample; and (iii) for each of one or more further predeterminedwavelength band(s) (each of which is sequentially selected using tunablefilter 140), image sensor 145 is thereafter used to simultaneouslydetect scattered or emitted photons from different locations on orwithin the sample. The outputs of detector 145 (for each of thewavelengths or wavelength bands selected by tunable filter 140 duringthe first time interval) are then combined by the computer (not shown)to generate the first chemical image of the sample.

During the second subsequent time interval: (i) sample 100 isilluminated with photons from source 110 to thereby produce photonsscattered or emitted by sample 100; (ii) image sensor 145 is then usedto simultaneously detect scattered or emitted photons in a firstpredetermined wavelength band (selected by tunable filter 140) fromdifferent locations on or within the sample; and (iii) for each of oneor more further predetermined wavelength band(s) (each of which issequentially selected using tunable filter 140), image sensor 145 isthereafter used to simultaneously detect scattered or emitted photonsfrom different locations on or within the sample. The outputs ofdetector 145 (for each of the wavelengths or wavelength bands selectedby tunable filter 140 during the first time interval) are then combinedby the computer (not shown) to generate the second chemical image of thesample.

Dynamic changes occurring in the sample between the first time intervaland the second time interval are next detected based on one or moredifferences between the first and second chemical images. Computeranalysis of the chemical image with or without the physical image may beused to detect (or enhance detection of) the dynamic changes. Thedynamic changes may also be detected by a user viewing a display of thechemical images.

In various embodiments, a series of many sequential chemical images areobtained rapidly in succession to generate a “movie” of the sample. Forexample, as many as 100 chemical images per second of the sample may beobtained in order to detect dynamic changes in the sample insubstantially real-time. In some embodiments, the temporal resolution ofthe chemical images in the sequence may be as fine a 1 millisecond,i.e., the system will generate a chemical image of the sample everymillisecond. Other temporal resolutions can also be selected including,for example, a temporal resolution that equates to chemical imagesspaced apart by as much as 15 minutes between adjacent images. Whenusing the present invention to monitor a particular process or reaction,the temporal resolution selected should be sufficient to detect dynamicchanges of interest that occur in the sample over time.

The present invention thus permits rapid observation of sample 100 withfull spatial information, and allows the monitoring of the evolution andchanges in sample 100 that are natural proceeding or occurring (i.e.,under equilibrium conditions), as well as those that are additionallyforced or imposed by creating a non-equilibrium condition via anexternal means (e.g., one or more external fields or forces applied tothe sample). In certain embodiments, the external means are applied to aspecific location within sample 100 (rather than the whole sample).Examples of samples that may be analyzed and observed used the dynamicchemical imaging techniques of the present invention includes biologicalsamples or micro-fluidic circuits undergoing changes over time. Thesechanges may include displacement, chemical interaction, a change inchemical state, phase change, growth, shrinkage, chemical decomposition,chemical metabolization and physical strain. Numerous other examples ofsamples/changes applicable to the present invention will be recognizedby the those skilled in the art and are considered within the scope ofthe present invention.

As noted above, the present invention may be used to detect dynamicchanges in the sample that result from application of an externalcondition to the sample. Such external conditions include, for example,varying an electric or magnetic field applied to or within sample 100between the first and second time intervals; varying an external opticalfield applied to or within the sample between the first and second timeintervals, wherein the external optical field is distinct from theoptical field initially used to illuminate the sample; varying theoptical field applied to or within the sample between the first andsecond time intervals, wherein the additional optical field is producedby pulsing the optical filed used to illuminate the sample; varyinginternally generated photons applied to or within the sample between thefirst and second time intervals; varying a polarization used toilluminate the sample between the first and second time intervals;varying a temperature of the sample between the first and second timeintervals; varying a pressure applied to the sample between the firstand second time intervals; or varying a stress applied to or within thesample between the first and second time intervals. In otherembodiments, a chemical gradient associated with the sample (e.g., achemical gradient imposed on the sample) varies between the first andsecond time intervals. In still further embodiments, a physiological orbiological stress is induced in the sample between the first and secondtime intervals.

In some embodiments, each chemical image in the sequence is made up ofmultiple separate spatially accurate wavelength-resolved images of thesample (each of which is formed from multiple “frames” as discussedabove), wherein each of the multiple separate spatially accuratewavelength-resolved images corresponds to one of a plurality ofdifferent depths within the sample. These embodiments are useful fordetecting chemical changes occurring throughout the volume of sample100, rather than changes occurring on a single surface or plane of thesample.

In still further embodiments, differences between or among variouschemical images in the sequence may be correlated (using, e.g., thecomputer discussed above or by a user) with orthogonal measurements ofthe sample made during each of the time intervals associated with thesequence, in order to enhance detection or observation of dynamicchanges in the sample. Examples of orthogonal measurements that may beused include measurements made using the following modalities: Ramanscattering, near infrared absorption (NIR), visual imagery, video orluminescence. Other orthogonal measurements may also be used and areconsidered to be within the scope of the present invention.

Finally, it will be appreciated by those skilled in the art that changescould be made to the embodiments described above without departing fromthe broad inventive concept thereof. It is understood, therefore, thatthis invention is not limited to the particular embodiments disclosed,but is intended to cover modifications within the spirit and scope ofthe present invention as defined in the appended claims.

1. A method for detecting dynamic changes that occur in a sample betweena first time interval and a second time interval using a series of atleast first and second sequential chemical images of the sample, whereinthe first chemical image corresponds to an image of the sample during afirst time interval, and the second chemical image corresponds to animage of the sample at a second time interval after the first timeinterval, the method comprising: (a) during the first time interval: (i)illuminating the sample with a plurality of photons to thereby producephotons scattered or emitted by the sample; (ii) using a two-dimensionalarray of detection elements to simultaneously detect scattered oremitted photons in a first predetermined wavelength band from differentlocations on or within the sample; and (iii) separately repeating step(a)(ii) for each of a plurality of further different predeterminedwavelength bands; (b) combining the results of steps (a)(ii) and(a)(iii) to generate the first chemical image of the sample; (c) duringthe second time interval: (i) illuminating the sample with a pluralityof photons to thereby produce photons scattered or emitted by thesample; (ii) using the two-dimensional array of detection elements tosimultaneously detect scattered or emitted photons in the firstpredetermined wavelength band from different locations on or within thesample; and (iii) separately repeating step (c)(ii) for each of theplurality of further different predetermined wavelength bands; (d)combining the results of steps (c)(ii) and (c)(iii) to generate thesecond chemical image of the sample; and (e) detecting dynamic changesoccurring in the sample between the first time interval and the secondtime interval based on one or more differences between the first andsecond chemical images.
 2. The method of claim 1, further comprisingvarying an electric field applied to or within the sample between thefirst and second time intervals.
 3. The method of claim 1, furthercomprising varying a magnetic field applied to or within the samplebetween the first and second time intervals.
 4. The method of claim 1,further comprising varying an external optical field applied to orwithin the sample between the first and second time intervals, whereinthe external optical field is distinct from an optical field used toilluminate the sample in step (a)(i).
 5. The method of claim 1, furthercomprising varying internally generated photons applied to or within thesample between the first and second time intervals.
 6. The method ofclaim 1, further comprising varying a polarization used to illuminatethe sample between the first and second time intervals.
 7. The method ofclaim 1, further comprising varying a temperature of the sample betweenthe first and second time intervals.
 8. The method of claim 1, furthercomprising varying a pressure applied to the sample between the firstand second time intervals.
 9. The method of claim 1, further comprisingvarying a stress applied to or within the sample between the first andsecond time intervals.
 10. The method of claim 1, wherein a chemicalgradient associated with the sample varies between the first and secondtime intervals.
 11. The method of claim 1, wherein a chemical gradientimposed on the sample varies between the first and second timeintervals.
 12. The method of claim 1, further comprising inducing aphysiological or biological stress in the sample between the first andsecond time intervals.
 13. The method of claim 1, further comprising:repeating steps (a)(ii) and (a)(iii) for each of a plurality of depthswithin the sample during the first time interval, wherein the firstchemical image of the sample includes a spatially accuratewavelength-resolved image chemical image associated with each of theplurality of depths within the sample; and repeating steps (c)(ii) and(c)(iii) for each of the plurality of depths within the sample duringthe second time interval, wherein the second chemical image of thesample includes a spatially accurate wavelength-resolved image chemicalimage associated with each of the plurality of depths within the sample.14. The method of claim 1, wherein step (e) further comprisescorrelating differences between the first and second chemical imageswith orthogonal measurements of the sample made during the first andsecond time intervals, wherein the orthogonal measurements correspond tomeasurements made using at least one of the following modalities: Ramanscattering, near infrared absorption (NIR), visual imagery, video orluminescence.
 15. A system for detecting dynamic changes that occur in asample between a first time interval and a second time interval using aseries of at least first and second sequential chemical images of thesample, wherein the first chemical image corresponds to an image of thesample during a first time interval, and the second chemical imagecorresponds to an image of the sample at a second time interval afterthe first time interval, comprising: (a) a source that illuminates thesample with a plurality of photons to thereby produce photons scatteredor emitted by the sample; (b) a two-dimensional array of detectionelements that, during the first time interval, (i) simultaneouslydetects scattered or emitted photons in a first predetermined wavelengthband from different locations on or within the sample, and (ii)thereafter, for each of a plurality of further different predeterminedwavelength bands, simultaneously detects scattered or emitted photonsfrom different locations on or within the sample; (c) a processor thatcombines the outputs of the two-dimensional array of detection elementsduring the first time interval to generate the first chemical image ofthe sample; (d) wherein, during the second time interval, thetwo-dimensional array of detection elements (i) simultaneously detectsscattered or emitted photons in the first predetermined wavelength bandfrom different locations on or within the sample, and (ii) thereafter,for each of the plurality of further different predetermined wavelengthbands, simultaneously detects scattered or emitted photons fromdifferent locations on or within the sample; (e) wherein the processorcombines the outputs of the two-dimensional array of detection elementsduring the second time interval to generate the second chemical image ofthe sample; and (f) wherein the processor detects dynamic changesoccurring in the sample between the first time interval and the secondtime interval based on one or more differences between the first andsecond chemical images.